EpiTox: A Multi-Modular Framework for Population-Aware Off-Target Prediction Highlighting MAGE-A3 Cross-Reactivity

Al-Hasani, H.; Ali, M.; Kraemer, S. D.; Heiss, K.; Sinclair, A.; Herz, T.; Meyer, P.; Hamde, P.; Roth, G.; Birkenfeld, J.; Selinger, O.; Schuster, S.

doi:10.64898/2025.12.10.693357

bioRxiv · 10.64898/2025.12.10.693357

EpiTox: A Multi-Modular Framework for Population-Aware Off-Target Prediction Highlighting MAGE-A3 Cross-Reactivity

Al-Hasani, H.·Ali, M.·Kraemer, S. D.·Heiss, K.·Sinclair, A.·Herz, T.·Meyer, P.·Hamde, P.·Roth, G.·Birkenfeld, J.·Selinger, O.·Schuster, S.

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Abstract

Targeting intracellular tumor antigens presented by peptide human leukocyte antigen (pHLA) complexes offers a promising immunotherapeutic strategy for patients with limited treatment options. However, development of pHLA-targeted drugs such as T-cell receptor (TCR) mimic antibodies (TCRm) and TCR-based therapeutics remains challenging due to severe offtumor and off-target toxicities arising from incomplete pHLA off-target profiling. The fatal neurological toxicities observed in the MAGEA3 TCR-T cell trial, potentially linked to unanticipated cross-reactivity with EPS8L2- and MAGEA12-derived peptides, underscore the urgent need for more comprehensive safety assessment. While current preclinical de-risking methods utilize sequence similarity searches, they lack the layered integration of genetic context, HLA binding profiles, and structured risk assessment needed to comprehensively evaluate peptide cross-reactivity. To address these limitations, we developed EpiTox, a computational multi-modular platform that systematically identifies and evaluates pHLA off-targets. EpiTox integrates proteome-wide sequence similarity, genetic context, HLA binding profiles, and layered risk assessment to provide a holistic evaluation of potential cross-reactive peptides. Using MAGEA3 as a model, EpiTox identified known toxic off-targets and predicted novel epitopes. Notably, an anti-MAGEA3 therapeutic TCRm antibody bound several newly predicted off-targets, including three SNP-derived peptides. By enabling comprehensive and predictive preclinical safety screening, Epi-Tox supports safer TCRm drug development and may help prevent future clinical failures.

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Al-Hasani, H., Ali, M., Kraemer, S. D., Heiss, K., Sinclair, A., Herz, T., Meyer, P., Hamde, P., Roth, G., Birkenfeld, J., Selinger, O., Schuster, S.. 2025-12-12. EpiTox: A Multi-Modular Framework for Population-Aware Off-Target Prediction Highlighting MAGE-A3 Cross-Reactivity. https://doi.org/10.64898/2025.12.10.693357

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